Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

The consequences of ubiquitous expression of the wingless gene in the Drosophila embryo.

The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.     

Engrailed gene expression in Drosophila imaginal discs.

Genetic and molecular analyses indicate that the Drosophila engrailed gene is required to distinguish posterior from anterior compartments in each segment of the developing animal. Here, the patterns of engrailed expression in the imaginal discs and ventral ganglion of Drosophila larvae are examined, using an antiserum against the engrailed protein and a novel image processing method to reduce non-specific background. As expected, engrailed expression generally is restricted to cells in the posterior compartment of the discs, and the patterns of expression allow refinements in the fate maps of the discs to be made. More significant is the finding that expression of the gene is highly variable in different regions of posterior compartments, suggesting that engrailed may do more than simply specify 'posteriorness'. In the ventral ganglion engrailed appears to be expressed by a subset of cells, primarily in the posterior regions of each segment. In wing discs from animals that are homozygous for the en1 mutation, the pattern of expression of the gene is altered, as opposed to being simply reduced uniformly in the posterior cells.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.     

Patterns of engrailed protein in early Drosophila embryos

By the onset of gastrulation during nuclear cycle 14 of Drosophila embryogenesis, the engrailed gene is expressed in fourteen one-cell-wide stripes. Each stripe defines the anlagen of the posterior compartment of a metameric segment. We report here several observations relating to the role and disposition of the engrailed protein during the embryonic stages that precede cellularization. We demonstrate that in embryos mutant for the engrailed gene, there were characteristic morphological abnormalities as early as the 6th cleavage cycle. In addition, the engrailed protein was detected in pre-cycle-9 embryos by Western blot analysis. When localization of engrailed protein begins during cycle 14, engrailed expression was first present in broad anterior and posterior regions before the fourteen-stripe pattern appeared.     

Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.     

A single cell analysis of engrailed expression in the early embryonic brain of the grasshopper Schistocerca gregaria: ontogeny and identity of the secondary headspot cells

The expression pattern of the segment polarity gene engrailed was studied at the single cell level in the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria, the neuromere containing the secondary headspot cells. The engrailed protein is first expressed in the protocerebrum at about 22% of embryogenesis by a group of identified neuroblasts bordering the antennal lobe. The number of immunoreactive neuroblasts increases up to 26% of embryogenesis and then rapidly declines so that by 30% only the three most posterior remain immunoreactive. These three neuroblasts become incorporated into the developing antennal lobe of the deutocerebrum. Subsequently, there is a progressive re-expression of the engrailed protein in the protocerebrum by the so-called six secondary headspot cells. These are the first born sibling progeny of three identified protocerebral neuroblasts which themselves expressed the engrailed protein prior to generating their lineages, and so represents a reacquisition of engrailed expression within identified clones. The secondary headspot cells are neurons which direct axonal processes into the developing optic tract and so contribute to the primary axon scaffold of the brain. From our analysis of their ontogeny, we conclude that the secondary headspot cells do not represent a segmental border in the brain.     

Embryonic expression of the engrailed homologue of Rhynchosciara americana

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.     

Establishment of segment polarity in the ectoderm of the leech Helobdella

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.     

Segmenting the fly embryo: logical analysis of the role of the segment polarity cross-regulatory module

Initially activated by the pair-rule genes, the expression patterns of the segment polarity genes engrailed and wingless become consolidated through inter-cellular interactions between juxtaposed cells. We delineate a logical model focusing on a dozen molecular components at the core of the regulatory network controlling this process. Our model leads to the following conclusions: (1) the pair-rule signals, which activate engrailed and wingless genes independently of each other, need to be operative until the inter-cellular circuit involving these two genes is functional. This implies that the pair-rule pattern is instrumental both in determining the activation of the genes engrailed and wingless in rows of adjacent cells, and in consolidating these expression patterns; (2) the consolidation of engrailed and wingless expression patterns requires the simultaneous activation of both autocrine and paracrine Wingless-pathways, and the Hedgehog pathway; (3) protein kinase A plays at least two roles through the phosphorylation of Cubitus interruptus, the effector molecule of the Hedgehog signalling pathway and (4) the roles of Sloppy-paired and Naked in the delineation of the engrailed and wingless expression domains are emphasized as being important for segmental boundary formation. Moreover, the application of an original computational method leads to the delineation of a subset of crucial regulatory circuits enabling the coexistence of specific expression states at the cellular level, as well as specific combination of cellular states inter-connected through Wingless and Hedgehog signalling. Finally, the simulation of altered expressions of segment polarity genes leads to results consistent with the published data.     

The expression of an engrailed protein during embryonic shell formation of the tusk-shell, Antalis entalis (Mollusca, Scaphopoda)

SUMMARY This study presents the first detailed account of the larval and early post-metamorphic development of a scaphopod species, Antalis entalis , since 1883. Special reference is given to the expression pattern of an engrailed protein during the formation of the embryonic (protoconch) and adult shell (teleoconch). We found that in the trochophore-like larva the engrailed protein is expressed in shell-secreting cells at the margin of the protoconch close to the mantle edge. During metamorphosis the growth of the protoconch and expression of the engrailed protein along its margin stop and the teleoconch starts to form. These data suggest a different genetic background regarding protoconch and teleoconch formation in the Scaphopoda and possibly all Conchifera, thus inferring a different evolutionary origin of both organs. The single anlage of the scaphopod protoconch contradicts earlier hypotheses of a monophyletic taxon Diasoma (Scaphopoda + Bivalvia), which has been mainly based on the assumption of a primarily bilobed shell in both taxa. Comparative data on engrailed expression patterns suggest nervous system patterning as the basic function of engrailed in the Bilateria. However, there are several independent gain-of-function events, namely segment compartmentation in the Annelida and Arthropoda, protoconch formation in the Mollusca, skeletogenesis in the Echinodermata, and limb formation in vertebrates. These findings provide further evidence that homologous genes may act in very different pathways of bilaterian body plan formation in various animal phyla.     

Allelic interactions at the engrailed locus of Drosophila: engrailed protein expression in imaginal discs

Many embryonic lethal engrailed (enlethal) mutations are known to partially complement the cuticular defects of the original engrailed mutation, en1. To explore the nature of this complementation, the adult phenotypes of several different en1/enlethal transheterozygotes were compared with the corresponding patterns of engrailed protein expression in third larval instar imaginal discs (determined by immunofluorescence). Transheterozygotes of en1 and deletions of the locus (enDf) typically show slight complementation in the adult cuticle. The pattern of engrailed protein expression in some en1/enDf wing discs is indistinguishable from en1 homozygotes, but in others the pattern is nearly normal. en1/enDf leg discs appear to express engrailed protein normally. Transheterozygotes of en1 and EMS-induced, cytologically normal enlethal alleles have almost normal adult cuticle phenotypes and also exhibit normal patterns of engrailed protein expression in all of the thoracic imaginal discs. Surprisingly, the intensity of anti-engrailed staining in these discs is elevated relative to that in wild type. en2 is an unusual lethal allele in that it does not complement either the en1 adult cuticle phenotype or the protein expression pattern in imaginal discs. Moreover, the cytologically normal enlethal alleles also complement en2, at least partially. Both wing and leg imaginal discs from en2/enlethal transheterozygotes show abnormal patterns of engrailed protein expression. These results are discussed in the context of an autoregulatory model for engrailed regulation.     

Segment polarity gene expression in a myriapod reveals conserved and diverged aspects of early head patterning in arthropods

Arthropods show two kinds of developmental mode. In the so-called long germ developmental mode (as exemplified by the fly Drosophila), all segments are formed almost simultaneously from a preexisting field of cells. In contrast, in the so-called short germ developmental mode (as exemplified by the vast majority of arthropods), only the anterior segments are patterned similarly as in Drosophila, and posterior segments are added in a single or double segmental periodicity from a posterior segment addition zone (SAZ). The addition of segments from the SAZ is controlled by dynamic waves of gene activity. Recent studies on a spider have revealed that a similar dynamic process, involving expression of the segment polarity gene (SPG) hedgehog (hh), is involved in the formation of the anterior head segments. The present study shows that in the myriapod Glomeris marginata the early expression of hh is also in a broad anterior domain, but this domain corresponds only to the ocular and antennal segment. It does not, like in spiders, represent expression in the posterior adjacent segment. In contrast, the anterior hh pattern is conserved in Glomeris and insects. All investigated myriapod SPGs and associated factors are expressed with delay in the premandibular (tritocerebral) segment. This delay is exclusively found in insects and myriapods, but not in chelicerates, crustaceans and onychophorans. Therefore, it may represent a synapomorphy uniting insects and myriapods (Atelocerata hypothesis), contradicting the leading opinion that suggests a sister relationship of crustaceans and insects (Pancrustacea hypothesis). In Glomeris embryos, the SPG engrailed is first expressed in the mandibular segment. This feature is conserved in representatives of all arthropod classes suggesting that the mandibular segment may have a special function in anterior patterning.     

The state of engrailed expression is not clonally transmitted during early Drosophila development.

In Drosophila embryos, boundaries of lineage restriction separate groups of cells, or compartments. Engrailed is essential for specification of the posterior compartment of each segment, and its expression is thought to mark this compartment. Using a new photo-activatable lineage tracer, we followed the progeny of single embryonic cells marked at the blastoderm stage. No clones straddled the anterior edges of engrailed stripes (the parasegment border). However, posterior cells of each stripe lose engrailed expression, producing mixed clones. We suggest that stable expression of engrailed by cells at the anterior edge of the stripe reflects, not cell-intrinsic mechanisms, but proximity to cells that produce Wingless, an extracellular signal needed for maintenance of engrailed expression. If control of posterior cell fate parallels control of engrailed expression, cell fate is initially responsive to cell environment and cell fate determination is a later event.     

Association of the Drosophila melanogaster engrailed protein with specific soluble nuclear protein complexes.

The Drosophila engrailed protein which contains a homeobox domain and specific DNA binding activity is believed to function in the regulation of gene expression during embryogenesis. Here we show that the engrailed protein interacts stably with specific complexes of soluble nuclear proteins when expressed artificially in a cell line and in the developing embryo. The engrailed complexes have molecular masses between 10(7) and 10(8) which suggests they contain a polymeric protein component. The complex is able to bind reversibly to DNA and a definitive purification shows it to be constituted of 12 distinct protein species, two of which are predominant. Purified, bacterially produced engrailed protein can be reconstituted with both culture cell and embryo nuclear protein fractions to form complexes of the same and related composition respectively. On the basis of these results we propose that protein--protein interactions as well as DNA binding are important for correct engrailed protein function in vivo.     

A Fragment of Engrailed Regulatory DNA Can Mediate Transvection of the White Gene in Drosophila

We have found a fragment of engrailed regulatory DNA that has an unusual effect on expression of a linked marker gene, white, in the P element transposon CaSpeR. Normally, flies homozygous for a given CaSpeR insertion have darker eyes than heterozygotes. However, when a particular engrailed DNA fragment is included in that transposon, homozygotes often have lighter eyes than heterozygotes. Thus, engrailed DNA appears to cause white expression to be repressed in homozygotes. The suppression of white is dependent on the proximity of the two transposons in the genome-either in cis (i.e., on the same chromosome) or in trans (i.e., on homologous chromosomes). Thus, the engrailed fragment is mediating a phenomenon similar to that mediated by the zeste gene at the white locus. However, the interactions we observe do not require, nor are influenced by, mutations of zeste. We suggest that the engrailed DNA contains one or more binding sites for a protein that facilitates interactions between transposons. The normal function of these sites may be to mediate interactions between distant cis-regulatory regions of engrailed, a large locus that extends over 70 kilobases.     

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system

BACKGROUND: The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS: We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS: The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.     

Barnacle duplicate engrailed genes: divergent expression patterns and evidence for a vestigial abdomen

SUMMARY Cirripedes (barnacles) are crustaceans that are characterized by a very peculiar body plan, in particular by the lack of an abdomen. To study their body plan, we searched for their engrailed gene. We found two engrailed ( en.a/en.b ) genes in cirripedes. The two engrailed genes of the rhizocephalan barnacle Sacculina carcini are expressed in the posterior compartment of developing segments and appendages. When the neuroectoderm differentiates into epidermis and neuroderm the expression patterns of en.a and en.b diverge dramatically. en.a expression fades in segment epidermis whereas it is subsequently detected ventrally in reiterated putative neural cells. At the same time, en.b expression increases in the epidermis, which makes it a very good segmentation marker. Five tiny en.b stripes are observed between the sixth thoracic segment and the telson. We interpret these stripes as the molecular definition of vestigial abdominal segments, being the remnant of an ancestral state in keeping with the bodyplan of maxillopod crustaceans. engrailed expression is the first molecular evidence for a segmented abdomen in barnacles.